Int J Nanomedicine. 2016; 11: 37893800.

Published online 2016 Aug 12. doi: 10.2147/IJN.S104166

PMCID: PMC4990392

Effects of silver nanoparticles in combination with antibiotics

on the resistant bacteria Acinetobacter baumannii
Guoqing Wan,1,2 Lingao Ruan,2,3 Yu Yin,2,3 Tian Yang,2,3 Mei Ge,2 and Xiaodong Cheng1,4

Author information Copyright and License information

Abstract
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Introduction
Drug-resistant Acinetobacter baumannii is an infectious pathogen that currently presents
serious clinical challenges. A. baumannii is particularly associated with hospital-acquired
infections such as pneumonia, bloodstream, abdominal, central nervous system, urinary
tract, and skin and soft tissue infections.1 A. baumannii can develop resistance against
antibiotics through several mechanisms;2 in particular, this bacterium is often resistant to
the carbapenems.3 Increasing numbers of carbapenem-resistant A. baumannii isolates have
been reported worldwide.4 The majority of such bacteria are extensively drug resistant,
which may include resistance to carbapenems and all other antibiotics except polymyxins
and tigecycline.5 Polymyxin B is effective against drug-resistant A. baumannii, but
systemic application carries risk of toxicity, primarily kidney toxicity and
neurotoxicity.6,7 A. baumannii infection is common in patients with severe infections, and
is often accompanied by other bacterial and/or fungal infections.8 Patients infected with
resistant A. baumannii have high mortality.9 Therefore, there is an urgent need to find
suitable therapeutic drugs for the treatment of resistant A. baumannii infections.
The ineffectiveness of synthetic antibiotics against drug-resistant bacteria has led to the
reemergence of interest in silver, which has an ancient history as an antibacterial agent.10
12 The antibacterial activity of silver nanoparticles (AgNPs) had been reported against
multiple species of bacteria; for example, Escherichia coli ATCC 8739,13 Staphylococcus
aureus ATCC1431,14 Escherichia fergusonii, and Klebsiella aerogenes ATCC
1950,15 among others. Synthesized AgNPs with capping agents, such as citrate, sodium
dodecyl sulfate, and polyvinylpyrrolidone show increased antibacterial activity against S.
aureus and E. coli.16 Huang et al reported antimicrobial activity against A. baumannii with
the synergistic combination of chitosan acetate and AgNPs.17 Jain et al studied the
interaction of AgNPs with commonly used antibiotics in Pseudomonas
aeruginosa,15 while Morones-Ramirez et al demonstrated that Ag+ treatment sensitized
Gram-negative bacteria to the Gram-positive-specific antibiotic vancomycin, both in vitro
and in vivo.18 However, the synergistic antimicrobial activity of antibiotics combined with
citrate-capped AgNPs has yet to be studied.
In the present study, we investigated the synergistic combinatorial effects of antibiotics
with AgNPs against drug-resistant A. baumannii obtained from clinical patients both in
vivo and in vitro. We also investigated the possible mechanisms of this synergistic effect.
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Materials and methods

Materials
Trisodium citrate, silver nitrate (AgNO3), and sodium borohydride (NaBH4) were used for
the synthesis of AgNPs. Rifampicin, tigecyline, polymyxin B (PMB), mucin, dimethyl
sulfoxide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), isopropyl-
-D-thiogalactoside, penicillinstreptomycin, and trypsinethylenediaminetetraacetic acid
were purchased from Sigma Aldrich (St Louis, MO, USA). A549 cells and HL-7702 cells
were purchased from the Type Culture Collection of the Chinese Academy of Sciences
(Shanghai, Peoples Republic of China). Roswell Park Memorial Institute-1640 medium
was purchased from Thermo Fisher Scientific (Waltham, MA, USA) and fetal bovine
serum was purchased from HyClone (Logan, UT, USA). No ethical committee approval
was required for this set of experiments because the experiments were performed on
commercially available cell lines and were considered exempt from full review by the
ethics Committee at Shanghai Jiao Tong University.
All animal procedures were approved by the Institutional Animal Care and Use Committee
at Shanghai Jiao Tong University. All the animal studies were performed according to the
Guiding Principles for the Care and Use of Laboratory Animals according to the
Regulations of the Peoples Republic of China for Administration of Laboratory Animals.
All animal procedures were approved by the Animal Ethics Committee of Shanghai Jiao
Tong University. C57BL/6 mice were purchased from Slac Laboratory Animal Co., Ltd.
(Shanghai, Peoples Republic of China).

Synthesis and characterization of AgNPs

In a three-necked round-bottomed flask, 20 mL trisodium citrate (1%) and 75 mL ultrapure
water were mixed for 15 minutes at 70C. To the solution, 1.5 mL of silver nitrate solution
(1%) was added; NaBH4 (1%) was then added, followed by rapid mixing. This mixed
solution was heated for 60 minutes, cooled to room temperature, and water was added to a
volume of 100 mL.
To characterize the morphology of the synthesized AgNPs, transmission electron
microscopy (TEM) analysis was performed using a Tecnai G2 Spirit 120 kV TEM
instrument (0.23 nm resolution) (FEI Company, Hillsboro, OR, USA).
AgNPs were further characterized by scanning the absorbance spectra in 300500 nm range
of wavelength with a multifunction full wavelength microplate analyzer (BioTek Co.,
Winooski, VT, USA).
A Malvern Zetasizer Nano-ZS instrument (Malvern, Louis, USA) was used to characterize
the zeta potential of the nanoparticles in the solution. Data were obtained and analyzed
using Zetasizer software (Malvern, Louis, USA).

Determination of minimum inhibitory concentration and fractional inhibitory

concentration
The bacterial strain A. baumannii (aba 1604; Fudan University Huashan Hospital,
Shanghai, Peoples Republic of China) was used as a model test strain to determine the
antibacterial activity of AgNPs. Various concentrations of AgNPs were incubated with
4105 bacteria in Luria Bertani (LB) broth medium in 96-well round-bottomed plates.
Bacteria were harvested at the indicated time points and the optical density of the samples
was assayed at 600 nm. All samples were plated in triplicate, and values were averaged
from three independent trials. The resistant A. baumannii strain (aba1604) was made from
clinical patients and following institutional ethical guidelines that were reviewed and
approved by the ethics committee at the Huashan hospital clinical ethics committee, Fudan
University. Consent from clinical patients was not deemed necessary by the Shanghai Laiya
centre for Biopharmaceutical R & D who obtained these strains for antibacterial drug
research and collected these samples, as the sample collection was part of normal patient
care.
To evaluate the antibacterial activity of AgNPs in combination with antibiotics, a two-
dimensional microdilution assay was used.19 Assays were carried out in LB broth growth
medium. Minimum inhibitory concentration (MIC) for each of the antibiotics was first
estimated, and the fractional inhibitory concentration (FIC) of a combination of antibiotics
and AgNPs was subsequently determined by the checkerboard microtitration method in a
96-well microtiter plate. Antibiotics and AgNPs were diluted to the following
concentrations (2MIC, 1MIC, 1/2MIC, 1/4MIC, 1/8MIC, 1/16MIC, and 1/32MIC) in the
two-dimensional microdilution assay.
The plates were incubated at 37C for 18 hours, and results were assayed by measuring the
optical density (OD)600. The combined antibiotic effect of agents A and B (where A is either
AgNO3 or AgNPs, and B is one of three antibiotic agents) was calculated as follows:

The FIC index:=MIC(A in combination with B)MIC(A alone)+MIC(B in combination with A)

MIC(B alone)

(1)
FIC index values above 4.0 indicate antagonistic effects, values between 0.5 and 4.0
indicate additive effects, and values lower than 0.5 indicate synergistic effects.20

Cytotoxicity assay
To determine the cytotoxic activity of the AgNPs on mammalian cells, A549 cells and HL-
7702 cells (1104cells/mL) were grown in Roswell Park Memorial Institute-1640 medium
containing 5% fetal bovine serum in a 96-well plate at 37C in an atmosphere of 5%
CO2 for 24 hours. Cells were treated with AgNPs, AgNO3, or control solutions at
concentrations ranging from 0.625 to 10 g/mL for another 24 hours. To determine the
viability, MTT (at a concentration of 0.1 mg/mL) was added to the wells and incubated for
4 hours at 37C and 5% CO2 to allow cell growth.21 In metabolically active cells, MTT
was reduced to an insoluble, dark purple formazan. The purple formazan was then
dissolved in dimethyl sulfoxide. The absorbance was measured at 570 nm using a
multifunction full wavelength microplate analyzer and readings were compared from
untreated cells. The OD values were used to sort out the percentage of viable cells by using
the following formula:

Percentageof viability=OD value of experimental sampleOD value of

experimentalcontrol (untreated)100
(2)

Assay for antimicrobial activity in vivo

Minimum lethal dose of AgNO3 or AgNPs in mice

Six-week-old male C57BL/6 mice (body weight ~20 g) were used for all animal
experiments. Mice were housed in a temperature- and humidity-controlled environment,
and had free access to food and water. Ten mice per group were given intraperitoneal
injections of 100 L total volume. Mice were treated as follows: no treatment, and 10, 20,
40, and 80 mg/kg AgNPs and AgNO3. Injected animals were observed for 3 days.

Determination of minimum lethal dose of A. baumannii for peritonitis mouse

model
Serial dilutions of A. baumannii ranging from 1107 to 11011 CFU, in 500 L sterile saline
supplemented with 8% mucin, were injected into the peritoneal cavity of mice.18 Animals
were observed for 2 days for determination of the survival rate.

Survival assays
Mice received intraperitoneal injections of the minimum lethal dose (MLD) of A.
baumannii, with a total volume of 500 L with 8% mucin. After 1 hour, ten mice in each
group received a 100 L intraperitoneal injection of either vehicle phosphate buffer saline
(PBS) or one of the different antibacterial treatments. Mice were observed for 2 days to
evaluate the survival rate.

Bacterial colonization assays

Surviving mice were euthanized and dissected, and their kidneys and lungs were collected.
These organs were ground under aseptic conditions, and the homogenates were dissolved in
sterilized saline water. These organ homogenates were then cultivated on LB plates at 37C
for 24 hours.

Cytokine profiling
Cytokine concentrations in the mouse plasma were measured at the indicated time after
infection by standard enzyme-linked immunosorbent assay kits following the
manufacturers instructions (Elabscience Biotechnology Co., Ltd, Wuhan, Peoples
Republic of China).

Antisense RNA models for detecting the synergistic mechanism of AgNPs

and antibiotic combinations
To investigate the pathways involved in the bacterial response to AgNPs, we conducted a
series of experiments in which the impacts of AgNPs and AgNO3 on different E.
coli antisense RNA-induced gene-silencing strains were examined.2224 The various gene-
silenced strains were arrayed in microwell plates, and then screened to determine how their
sensitivity to each of the different Ag formulations compared to the parent strain. The gene-
silenced E. coli strains were treated with isopropyl--D-thiogalactoside at appropriate
concentrations (Table 1), and portions of the culture were transferred into 96-well plates.
Sublethal concentrations of AgNPs, AgNO3, rifampicin, tigecyline, and PMB were added to
the gene-silenced bacteria in 96-well plates. Finally, the plates were incubated at 37C for
16 hours and shaken at 80 rpm. Absorbance was measured at 600 nm using a multifunction
full wavelength microplate analyzer, and OD values were used to calculate the inhibition
ratio (I) by using the following formula:

I(%)=OD value of control(untreated)OD value of sampleOD value of control(untreated)100

(3)

I(%)=Iantisense RNA-induced gene-silencing strainsIE.coliDH5/pHN678

(4)

Table 1
The optimal concentration of IPTG for antisense RNA strains

Statistical analysis
Each assay was repeated three times. Data are presented as mean standard deviation,
unless otherwise noted. Comparisons between multiple groups were made using one-way
analysis of variance (ANOVA) and all analyses were performed using SPSS 21.0 statistical
software (21.0; IBM Corporation, Armonk, NY, USA). The threshold for statistical
significance was set at P<0.05.
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Results

Characterization of AgNPs
Based on the image in Figure 1A, AgNPs stabilized by citrate had good dispersion. The
particle size distribution was shown by counting AgNPs particle numbers based on TEM
images and the histogram of particle size distribution was in Figure 1B. AgNPs were 512
nm in diameter, with an average size of 8.4 nm. The nanoparticles were found to be stable
for over 6 months, even at 37C. The zeta potential of the synthesized AgNPs is
summarized in Figure 1C. The ultravioletvisible (UVvis) spectra of the solution samples
are reported in Figure 1D. A single strong peak was observed at 392 nm, which indicates
the synthesis of spherical nanoparticles. In practice, dispersion was stable if the zeta
potential was higher than 30 mV or less than 30 mV. Supporting the earlier statement, we
observed that AgNPs dispersed in water were highly stable with a zeta potential value of
44.5 mV.

Figure 1
Appearance and physicochemical characteristics of AgNPs.

Antibacterial activities of AgNP combination treatments

Combination antibiotic therapy is a strategy often employed in the treatment of multiple
drug resistance (MDR) A. baumannii. Because PMB, rifampicin, and tigecycline are all
commonly used against MDR A. baumannii in combination with other antibiotics,2527 we
selected these three antibiotics to evaluate potential combinatorial effects with AgNPs. It
was seen that AgNPs displayed potent antimicrobial activity against A. baumannii, with an
MIC of approximately 2.5 g/mL, similar to that of AgNO3. Typical drug MICs,
summarized in Table 2, were 0.25 g/mL for PMB, 3.12 g/mL for rifampicin, and 3.12
g/mL for tigecycline.

Table 2
FIC index of combinations among silver and antibiotics against Acinetobacter baumannii
The FIC of AgNPs and the various antibiotic combinations were investigated and are
summarized in Table 2. These experiments showed that PMB and rifampicin acted
synergistically (P<0.5) with AgNPs and AgNO3, while tigecycline did not show synergy
(P>0.5) with either AgNPs or AgNO3.
Cytotoxicity of AgNPs in vitro
Many investigations have reported on the inhibitory effects of AgNPs on cells. For
example, Beer et al found that AgNPs inhibited the proliferation of A549 cells in a dose-
dependent manner,28 whereas Foldbjerg et al reported that AgNPs induced increase in
reactive oxygen species (ROS) level in A549 cells.29 Here, we used the method described
by Foldbjerg et al to evaluate the cytotoxicity of AgNPs.29
As shown in Figure 2, high concentrations of AgNO3 significantly affected cell growth. By
comparison, exposure to AgNPs at a higher concentration of 10 g/mL did not exhibit
significant cytotoxicity in A549 and HL-7702 cells. These results demonstrate that the
cytotoxicity of AgNPs is lower than that of AgNO3.

Figure 2
Relative survival of A549 and HL-7720 cells exposed to AgNPs.

Effects of AgNPs on antimicrobial activity in vivo

The acute toxicity of AgNPs and AgNO3 were measured in vivo to establish the median
lethal dose (LD50) (Figure 3). The LD50 of AgNPs was determined to be between 20 and 40
mg/kg, and the LD50 of AgNO3was also found to be between 20 and 40 mg/kg. These
LD50 values for AgNO3 are similar to those reported in an earlier toxicity study.18

Figure 3
Toxicity of AgNPs and AgNO3 in mice.
Infection was established in mice through intraperitoneal delivery of 5109 A.
baumannii cells suspended in an aqueous solution containing 8% mucin. Within 24 hours
after the time of injection, all infected mice had died. Thus, A. baumannii was used at this
same concentration of 5109 cells for all subsequent experiments.
The PMB and AgNPs combination and the PMB and AgNO3 combination showed the same
synergistic antibiotic effects that we had earlier observed in vivo (Figure 4). We observed
the mice living condition for 1 week, and calculated 2 days survival rate. A mixture of
AgNO3 and PMB (3 mg/kg and 10 g/kg, respectively) resulted in a survival rate of 40%.
When either of these two compounds was given alone as a single-dose treatment, survival
rates were 0%. By comparison, even a low-dosage mixture of AgNPs and PMB (2 mg/kg
and 10 g/kg, respectively) resulted in a high survival rate of 60%.
Figure 4
Survival of mice given AgNPs or AgNO3 with PMB in a peritonitis infection model.
Two days after administration in mice, we dissected a portion of mice for bacterial
colonization assay. Representative images of bacterial growth are presented in Figure 5,
which show that AgNO3 and AgNPs enhanced the action of PMB against A. baumannii in
peritonitis infection model. Figure 5A and B shows that there are plenty of bacteria present
in both the kidney and lungs when animals are treated with PMB alone at a dose of 250
g/kg. With the addition of AgNO3 (3 mg/kg, 18 M) or AgNPs (2 mg/kg, 18 M), no
bacteria were detected in the kidneys or lungs. When we reduced the PMB dose to 50
g/kg, we found that the kidney and lung tissues contained only a small amount of bacteria.
Further reduction of the PMB dose to 10 g/kg was less effective, leaving a substantial
bacterial burden in the kidney and lungs. Moreover, we also tested the blood and ascites of
infected mice and found that they did not contain bacteria under the combined
administration of PMB (50 g/kg) with either AgNO3 (3 mg/kg, 18 M) or AgNPs (2
mg/kg, 18 M) (Figure 5C and D). These results indicate not only that AgNO3 can enhance
the antibacterial activity of antibiotics but also that AgNPs possess antibacterial capability
in vivo.

Figure 5
Bacterial burdens in Acinetobacter baumannii-infected mice after treatment with PMB
combined with AgNO3 or AgNPs.
To analyze whether AgNO3 or AgNPs combined with antibiotics regulate inflammation
during A. baumannii infection, we assessed proinflammatory cytokines in the mouse
plasma using enzyme-linked immunosorbent assay. The levels of tumor necrosis factor
alpha (TNF-) and interleukin (IL)-6 decreased significantly in the mouse plasma of treated
mice compared to those of model mice at 18 hours post infection (Figure 6). AgNO3 and
AgNPs could enhance the action of PMB against A. baumannii in vivo.

Figure 6
AgNO3 or AgNPs combined with antibiotics-modulated Acinetobacter baumannii-induced
inflammatory reaction.
Mechanisms for enhanced antimicrobial activity of AgNPs in combination
with antibiotics
To better understand the mechanisms supporting the synergistic effects of AgNPs
combined with antibiotics, we used antisense RNA-induced gene silencing to silence the
expression of a number of genes in E. coli (Figure 7). The difference in inhibition rate
between a set of gene-silenced E. coli strains and the control E. coli DH5/pHN678 strain
is represented by color in our heat map analysis; dark red represents the most sensitive
strains, whereas dark blue represents the least sensitive strains, relative to the parent strain.
We found that silencing of rpoD, kdsA, kdsB, lpxC, and mutL resulted in sensitivity to both
AgNO3 and AgNPs. Silencing of rpsR, rpsL, rpsA, murB, murA, leuS, and dnaB led only to
sensitivity to AgNO3, which indicates that AgNPs act at a higher selectivity than AgNO3. In
addition, the rpoD-silenced strain was sensitive to rifampicin, while the silencing of kdsA,
kdsB, and lpxC increased sensitivity to PMB.

Figure 7
Heat map analysis of sensitivity to AgNPs and AgNO3 in gene-silenced Escherichia
coli strains relative to the control E. coli DH5/pHN678 strain.
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Discussion
A. baumannii has been proved to be resistant to many kinds of antibiotics, which is suitable
for genetic exchange. Fournier et al reported an 86 kb genomic region naming AbaR1
resistance island in AYE which had 45 resistance genes in the MDR isolate.30 The key
resistance genes were those coding for AmpC, VEB-1, and OXA-10 beta-lactamases,
tetracycline efflux pumps, and various aminoglycoside-modifying enzymes. Moreover,
Fournier et al found that 17 out of the 22 clinical A. baumannii isolates showed an original
ATPase ORF. These 17 isolates contained eleven isolates that are resistant to several
antibiotic families, including -lactams, and six other isolates susceptible to -lactams.
AgNPs could damage the membrane potential, prevent ATP production, increase the level
of ROS, and damage the membrane lipids as well as DNA, which demonstrated that AgNPs
have broad-range antibacterial properties, including A. baumannii.31
It has been reported that citrate-capped AgNPs are less toxic to mammalian cells and show
increased antimicrobial activity against S. aureus and P. aeruginosa.32 Our results support
these findings, and further build from them to explore the toxicity of AgNPs to A549 and
HL-7702 cells, and to describe the effects of AgNPs on drug-resistant A. baumannii. Our
study demonstrates that the cytotoxicity of AgNPs is lower than that of AgNO3.
The combination of AgNPs and either PMB or rifampicin showed strong synergistic
antimicrobial effects. Moreover, the pairing of AgNPs and PMB showed an enhanced effect
against A. baumannii in vivo, which suggests the possibility of a clinical application for this
combination therapy.33
IL-6 and TNF- are cytokines that have been shown to play an important role in the host
immune response against intracellular pathogens in murine models. Smani et al
demonstrated that A. baumannii induced the release of TNF- and IL-6 and increased the
Ca2+ influx.34 The levels of TNF- and IL-6 decreased significantly in the mouse plasma of
treated mice compared to those of model mice, which further proved that inhibiting
proinflammatory signals could be protective during A. baumannii infections. Sarkar et al
reported that AgNPs could modulate human macrophage responses to Mycobacterium
tuberculosis.35 A. baumannii is also highly correlated with the host immune status.35 We
have verified the role of AgNPs on A. baumannii. Hence, we hypothesized that AgNPs
could modulate human macrophage responses to A. baumannii, as the mode of M.
tuberculosis.
E. coli is one of the most representative model organisms in experimental biology and
medical study; there are a large number of experimental studies on the drug mechanisms
and targets using E. coli.2224 On the one hand, A. baumannii is a common clinical
pathogen, which has received increased attention.36 A. baumannii and E. coli are Gram-
negative bacteria, and they have similar cell structure.37 So we probe the mechanism of
synergistic effect of AgNPs and antibiotics combination against A. baumannii using E.
coli as a model system.
The probable role of PMB in such drug synergy is its rapid permeabilization of the outer
cell membrane, allowing enhanced penetration by AgNPs. Polymyxin B can displace
Mg2+ or Ca2+, and also binds to the Lipid A component of lipopolysaccharide (LPS),
resulting in changes to the outer membranes of bacteria. Our gene-silencing experiments
also suggested that LpxC plays a role in sensitivity toward AgNPs and AgNO3. Lin et al
found that an LpxC inhibitor blocked the ability of bacteria to activate the sepsis cascade,
enhanced opsonophagocytic killing of A. baumannii, and protected mice from lethal
infection.38 Moreover, the potential contributions of PMB and AgNPs both involve Lipid
A of LPS, a convergence which supports their synergistic effects.
KDO 2-keto-3-deoxyoctanoic acid (KDO) plays an essential role in LPS biosynthesis, and
may serve a universal role in group 2 capsule biosynthesis by linking the polysaccharide
region to the lipid domain.39 The kdsA gene encodes the protein KDO 8-phosphate
synthetase, which catalyzes the first step of the KDO synthetic reaction.40 The kdsB gene
encodes CMP-KDO synthetase, which is essential to the LPS biosynthesis pathway.41 Our
gene-silencing results suggested that kdsA and kdsB are involved in bacterial sensitivity
toward AgNPs and AgNO3, as well as PMB.
Similarly, we observed that silencing of RpoD, which is involved in promoter localization
and plays a crucial role in transcription initiation,42 increased the sensitivity of E.
coli toward AgNPs and AgNO3. Most of the factors investigated belong to the 70 family,
and all bacteria express one or more 70 factors. The 70 factor sequence is highly
conserved and plays an important role in bacterial growth. Rifampicin has a molecular
mechanism of action that involves inhibition of DNA-dependent RNA polymerase.43 In E.
coli, this enzyme is a complex oligomer comprising four subunits: , , , and , encoded,
respectively, by rpoA, rpoB, rpoC, and rpoD, and their disruption interferes in the
transcription process.43 The potential mechanisms of both rifampicin and AgNPs involve
effects on DNA-dependent RNA polymerase, which is the evidence that supports their
synergistic effects (Figure 8).

Figure 8
Proposed mechanisms of the combination of AgNPs/Ag+ with antibiotics against
G+ negative bacterium.
Silver and silver-containing compounds have recently drawn increasing interest as
antimicrobial agents for treating bacterial infections. AgNPs showed synergy of
inhibiting P. aeruginosa biofilms when combined with sub-MIC levels of
aztreonam.44 Combination of AgNPs with ceftazidime also showed a synergy to inhibit P.
aeruginosa.45 AgNPs prepared as described by Tiwari et al exhibited tremendous
antibacterial activity against a carbapenem-resistant strain of A. baumannii.3 This required
an efficient treatment regimen, and the combination of rifampin with imipenem had been
evaluated in clinical infections caused by a highly imipenem-resistant A.
baumannii strain.46 Yoon et al showed that the combination of PMB plus imipenem was as
effective as PMB plus rifampin against a carbapenem-resistant strain of A. bau-
mannii.26 Therefore, drug treatment with newer antimicrobials or antimicrobial
combinations has become increasingly important to eradicate these infections. According to
our study, the combination of AgNPs with antibiotics could be an effective solution to the
problem of carbapenem-resistant strains of A. baumannii, potentially at lower and less-toxic
doses than what is now typically used clinically.
Investigators have previously suggested that combination drug therapy could be an
effective tool to prevent the emergence of bacterial resistance, especially for patients
infected with Gram-negative bacteria that have developed resistance to a single
therapy.47 Our study showed the synergistic effects of combining AgNPs and PMB or
AgNPs and rifampicin against drug-resistant A. baumannii isolated from clinical patients.
Considering the lower toxicity of AgNPs compared to other treatment options, these drug
combinations have potential as useful tools for the clinic.
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Acknowledgments
The authors are very grateful to Professor Zhu Demei from Fudan University Huashan
Hospital, who provided the resistant A. baumannii strain (aba1604) from clinical patients.
This work was funded by The National Major Scientific and Technological Special Project
for Significant New Drugs Development (2012ZX09301002-003-007).
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Footnotes
Disclosure

The authors report no conflicts of interest in this work.

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